Spatially resolved ligand-receptor binding assays

ABSTRACT

A method for analyzing the results of a ligand-receptor binding assay comprising the steps of:(a) providing the results of a ligand-receptor binding assay; and(b) qualifying the results of a ligand-receptor binding assay.More particularly, the ligand-receptor binding assay involves the steps of combining appropriate reagents in which receptors attached to a solid support, a sample suspected of containing a ligand, and a conjugate comprising a label form a complex in which the label is present at a concentration that is directly proportional to the amount of ligand present in the sample. Alternatively, the ligand-receptor binding assay involves the steps of combining appropriate reagents to perform a ligand-receptor binding assay in which receptors attached to a solid support, a sample suspected of containing a ligand, and a conjugate comprising a label form a complex in which the label is present at a concentration that is inversely proportional to the amount of analyte present in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is application is a continuation of copending U.S. patentapplication Ser. No. 13/153,934, filed on Jun. 6, 2011.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates to determination of the concentration of a ligandin a sample, more particularly, a ligand that specifically binds to areceptor.

2. Discussion of the Art

For the past several decades, immunoassays have been performed usingfluorescence, chemiluminescence, or other means of generating a signalin response to an analyte. Currently, many immunoassays are performed bymeasurement of the intensity of a light signal generated in the totalvolume of a reaction mixture. The light signal generated can be measuredby an optical means, wherein the light signal generated is emitted by alarge number of molecules. In a typical embodiment, these immunoassayscan be carried out by combining a sample suspected of containing anantigen with a reagent comprising a first antibody attached to a solidsupport, e.g., a bead, a microparticle, to form a reaction mixture. Theantigen, if present in the sample, specifically binds to the firstantibody. A conjugate, which comprises a second antibody having a labelattached thereto, is introduced to the reaction mixture and specificallybinds to the antigen, which is specifically bound to the first antibody,which, as stated previously, is attached to the solid support. Such animmunoassay is referred to as a sandwich immunoassay or an immunometricassay. This type of immunoassay is shown schematically in FIG. 1. Thesignal attributable to the label is then measured after unboundconjugate is removed from the reaction mixture, typically by performinga wash step. The signal that is derived from the total volume of thereaction mixture is measured and then compared to a calibration curve toestablish the concentration of antigen present in the sample.

Another type of immunoassay is called a competitive immunoassay. In atypical embodiment, an unlabeled antigen and a labeled antigen competefor the same antibody site. Alternatively, an antibody and a labeledantibody compete for the same antigen site. In an example of the former,a labeled antigen and an unlabeled antigen are used. A solid support iscoated with an antibody that can specifically bind to either the labeledantigen or to the unlabeled antigen. The solid support, the labeledantigen, and a patient's sample suspected of containing the antigen arecombined. Of course, any antigen in the patient's sample is unlabeled.The labeled antigen and the unlabeled antigen compete for antibody siteson the solid support. Only when the labeled antigen attaches to theantibody on the solid support can a signal be produced, because only thelabeled antigen can generate a signal. The amount of antigen in thepatient's sample is inversely proportional to the amount of signalproduced. This type of immunoassay is shown schematically in FIG. 2.

In performing immunoassays using different optical methods, a number ofparameters must be considered. These parameters include the timerequired to perform the immunoassay, the amount of sample needed tocarry out the immunoassay, the amount of additional reagents needed tocarry out the immunoassay, the number of steps needed to complete theimmunoassay, the sensitivity of the immunoassay, and the dynamic rangeof the immunoassay. The dynamic range can often cover three or moreorders of magnitude. For many decades, immunoassays that utilizemagnetic microparticles have been shown to provide adequate values formost of the parameters mentioned previously. Magnetic microparticlesallow separation of analyte bound to conjugate from unbound conjugateand other reagents in a simple manner. Another attractive property ofmagnetic microparticles is that they can easily be controlled in asolution in order to allow for binding of analyte or conjugate in thesolid phase in a relatively short time. By making use of magneticattraction, magnetic microparticles can be moved and washed in order toprovide information about only the analyte bound to the magneticmicroparticles.

A major drawback with the use of any microparticle as the solid supportis lack of uniformity from microparticle to microparticle with respectto the amount of antibody coated on the microparticle. Another drawbackis the undesired interaction of the conjugate with the microparticles.Such undesired interaction may affect results of the immunoassay and,consequently, may require extensive study of a number of differentmicroparticles, made by different manufacturers, for use on animmunoassay analyzer. Another drawback presents itself when immunoassaysare performed in a reaction vessel. The conjugate can bind to thesurfaces of the reaction vessel, which is undesirable. These drawbacksnot only limit sensitivity of an immunoassay, but can yield falseresults upon measurement of the analyte.

Additional problems that may arise in immunoassays involve (a)non-specific binding of the conjugate to the solid support and (b)aggregation of reagents. These problems are a major concern todevelopers of an assay. Typically, methods to reduce non-specificbinding involve not only tailoring of reagents, but also mixing them inappropriate proportions to provide the desired results. These methods,which entail a significant amount of trial and error, often result inmaking development of an assay a long process, as well as makingdevelopment of an assay an empirical process, with the result thatreagents often vary from one lot to another lot. Moreover, a signalresulting from non-specific binding of the conjugate can be higher thanthe signal resulting from specific binding of the conjugate to ananalyte, thereby limiting the sensitivity of the immunoassay. Onlythrough the use of calibration using samples free of any analyte can theeffects of non-specific binding during an actual assay be estimated.

Another potential drawback of a typical immunoassay is that after theassay is performed, the only record of the assay is the value of signal.There is no opportunity to recheck the sample for defects or to obtainmore information if new methods of analysis become available. Not onlywill there be no record of the properties of the solid phase, there willalso be no way to review recorded data using a newly developedalgorithm. The ability to use historical data in order to extract newinformation is not possible.

Therefore, a need exists to develop analytical instruments and methodsfor addressing non-specific binding and undesired performance of thesolid phase in a given assay while simultaneously acquiring data fromthe assay to improve sensitivity of the assay. A need exists to reduceuse of reagents and provide measurement in an adequate time for use inboth a laboratory setting and a point-of-care setting. In principle,such a method would also provide real-time quality control as the assayis being performed. Furthermore, it is desired to alleviate the need togenerate new calibration curves and to reduce the variation of reagentsfrom lot to lot. It would also be desirable to maintain a record of theassay in such a manner that it can be reviewed at a later date throughthe use of different methods as these methods become available.

New detection methods can be coupled with devices from the emergingfields of nanotechnology and microfluidics to provide smaller, moreeffective, and more sensitive assays for detecting and measuringanalytes in biological samples.

SUMMARY OF THE INVENTION

This invention provides a method for analyzing a ligand-receptor bindingassay comprising the steps of:

-   -   (a) qualifying at least one signal of a ligand-receptor binding        assay; and    -   (b) providing the results of the ligand-receptor binding assay.

More particularly, the ligand-receptor binding assay involves combiningappropriate reagents in which receptors attached to a solid support,such as, for example, a microparticle, a sample suspected of containinga ligand, such as, for example, an analyte, and a conjugate comprising alabel form a complex in which the label is present at a concentrationthat is directly proportional to the amount of ligand present in thesample. The label of the conjugate is attached to a second receptor,which is different from the receptor attached to the solid support.Alternatively, the ligand-receptor binding assay involves combiningappropriate reagents to perform a ligand-receptor binding assay in whichreceptors attached to a solid support, such as, for example, amicroparticle, a sample suspected of containing a ligand, such as, forexample, an analyte, and a conjugate comprising a label form a complexin which the label is present at a concentration that is inverselyproportional to the amount of analyte present in the sample. The labelof the conjugate is attached to a ligand, which is the same ligand asthe ligand suspected of being in the sample. In order to improve thesensitivity of the immunoassay, an optional reaction step and anoptional washing step can be employed to reduce non-specific binding andremove any excess of conjugate. Another alternative is a one-stephomogeneous immunoassay, which does not require a separation step.

In one embodiment, microparticles bearing receptors on the surfacethereof, a sample suspected of containing an analyte, i.e., the ligand,and a conjugate comprising a label attached to a second receptor, inwhich the second receptor differs from those receptors attached to themicroparticles, are introduced into a reaction vessel and allowed toreact, whereby a complex comprising a label that emits a light signal isformed. The reaction vessel is capable of allowing the complexescomprising the labels that emit light signals to be recorded in animage. The resulting image is capable of being stored for use at a latertime. The light signal from the image is qualified before being used asa measurement of the concentration of an analyte. The image includes anumber of pixels, and the image is qualified and quantified on apixel-by-pixel basis.

Qualification involves restricting the analysis to those portions of theimage where complexes are located and measuring the value of intensityof light emanating from complexes in the image. An algorithm that issuitable for qualifying a light signal from a complex in an imagecomprises the steps of:

-   -   (a) acquiring a first fluorescence image to determine the        location of a first conjugate in the complex;    -   (b) selecting pixels in the image for analysis;    -   (c) calculating and recording the average and variance of counts        per pixel for the pixels selected in step (b);    -   (d) omitting from the analysis pixels that have counts greater        than or less than a specified level of variance; and    -   (e) calculating the average of counts per pixel of the remaining        pixels.        From the data obtained in step (e), the concentration of an        analyte in a sample can be determined.

In an optional step, a white light image of the reaction mixture can beobtained to determine the location of complexes attached to a solidsupport. In another optional step, a second fluorescence image can beacquired to determine the location of a second conjugate in the complex.The second fluorescence image can be used to increase sensitivity withrespect to determining the concentration of an analyte in a sample.

With respect to sandwich immunoassays, the receptor attached to thesolid support is often referred to as a capture antibody. The secondreceptor, which is attached to the label is often referred to as adetection antibody. The reaction vessel can be a micro-well of amicro-well plate. In a typical sandwich immunoassay, after the reactionmixture is allowed to incubate for a prescribed period of time, thereaction mixture is typically washed to remove any excess of detectionantibody and other unbound substances. The complex that remains in thereaction vessel is then imaged by, for example, a fluorescencemicroscope equipped with a digital camera. The average value of lightintensity per pixel is then determined. The value thus determined canthen be used to determine the concentration of antigen in the sample.

With respect to certain types of competitive immunoassays, the receptorattached to the solid support can also be deemed a capture antibody.However, when an antigen attached to a label, and the sample issuspected of containing the antigen, this particular type of competitiveimmunoassay does not have a detection antibody. The reaction vessel canbe a micro-well of a micro-well plate. In this particular type ofcompetitive immunoassay, after the reaction mixture is allowed toincubate for a prescribed period of time, the reaction mixture istypically washed to remove the excess of labeled antigen and any othersubstances remaining in the reaction mixture. The material remaining inthe reaction vessel is then imaged by, for example, a fluorescencemicroscope equipped with a digital camera. The average value of lightintensity per pixel is then determined. The value determined is thenused to determine the concentration of antigen in a sample. As indicatedpreviously, there are other types of immunoassays that can be carriedout to provide the results needed to carry out the imaging aspects ofthis invention.

In another embodiment, the ligand can be single strand of nucleic acid,e.g., DNA, RNA, and the receptor can be complementary strand of nucleicacid, e.g., DNA, RNA. In this embodiment, the ligand-receptor bindingreaction can be used to identify the presence of a gene or specificnucleic acid sequence, e.g., DNA sequence, RNA sequence.

An alternative method for providing additional information relating tothe aforementioned ligand-receptor binding assays involves counting ofthe number of microparticles or fluorescent spots that meet or exceed atleast one selected criterion for calculation of intensity. Thisalternative method can provide an internal control for indicating properperformance of the assay. In principle, comparison of results from assayto assay can be performed to ensure that each assay is reliable, i.e.,sufficiently sensitive and sufficiently precise. The average value offluorescence intensity per microparticle can be used to determine theconcentration of a ligand, in contrast to the value of intensity perpixel. The two values should both agree with values from the calibrator,thereby yielding two statistical measures that can be used to determineconcentration of an analyte.

The method described herein also enables maintaining a record of theassay in a manner similar to those in which x-rays and tissue pathologyrecords are maintained. Because images are acquired and stored off-line,i.e., by way of computer data storage that is not available forimmediate use on demand by the system without human intervention,different procedures for analysis can be employed at a later date. Abenefit of this feature is the enablement of direct comparison of assayresults from samples taken from the same patient at different times. Inthis manner, information about an actual sample and assays thereof isnot lost and can be shared or reviewed at a later time.

The method described herein addresses problems inherent in measurementof total signal by generating fluorescence images of complexescomprising microparticles after a ligand-receptor binding assay isperformed. In the method described herein, measurement of intensity oflight emitted from the total volume of a sample is replaced by analysisof images of the complex comprising an analyte from the sample in orderto improve the sensitivity of the assay. By incorporating spatialinformation that is not contained in conventional ligand-receptorbinding assays, aggregation of reagents and non-specific binding can beeliminated from the signal generated and the analyte in the sample canbe qualified and simultaneously or subsequently quantified. For example,with respect to qualification, undesired artifacts associated with asolid phase, such as, for example, microparticles, can be examined andremoved before the use of intensity information. Alternatively, if it isdesired that the degree of aggregation of proteins be measured, theaforementioned spatial information relating to aggregation can bemeasured, rather than removed from the signal generated. For example,aggregation of amyloids is an indicator of Alzheimer's disease.Furthermore, with respect to quantification, the method described hereinallows the omission of intensity information from reagents andconjugates that have non-specifically bound to the wall of a reactionvessel and from reagents and conjugates diffusing in the reactionmixture. Spatial information can be used to precisely define the regionin an image from which intensity should be measured in order to performa precise and accurate ligand-receptor binding assay.

Conventional assays require a large quantity of sample and a largequantity of reagent to provide sufficient signal. The method describedherein requires only a small amount of a solid support material, suchas, for example, only a few hundred coated microparticles, e.g., up toabout two hundred (200) microparticles, or fewer. The method describedherein enables the rechecking of samples for defects or to obtain moreinformation if new methods of analysis become available.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating a sandwich immunoassay.

FIG. 2 is a schematic diagram illustrating a competitive immunoassay.

FIG. 3 is a fluorescence image of a capture antibody labeled with a redfluorophore.

FIG. 4 is a fluorescence image of a detection antibody attached to amicroparticle.

FIG. 5 is an image showing regions of interest from which the averageintensity per pixel can be calculated.

FIG. 6 is a white light image of microparticles having complexes of anantibody:analyte:conjugate (i.e., monoclonal antibody19C7:troponin:conjugate M06-PE) attached thereto.

FIG. 7 is a fluorescence image of microparticles having complexes of anantibody:analyte:conjugate (i.e., monoclonal antibody19C7:troponin:conjugate M06-PE) attached thereto.

FIG. 8 is an image of the region of interest based on the images of FIG.6 and FIG. 7. FIG. 8 was generated by a software program.

FIG. 9 is an overlay of the fluorescence image of FIG. 7 on the image ofthe region of interest of FIG. 8.

FIG. 10 is a calibration curve showing the increase in sensitivity of animmunoassay resulting from the use of an imaging algorithm describedherein. For this calibration curve, the calibrators of the analyte(i.e., troponin) range from 10 pg/mL to 50,000 pg/mL.

FIG. 11 is a calibration curve showing the increase in sensitivity of animmunoassay resulting from the use of an imaging algorithm describedherein. For this calibration curve, the calibrators of the analyte(i.e., troponin) range from 0 pg/mL to 200 pg/mL. In FIG. 11, theaverage value of fluorescence intensity per pixel from the total area ofthe image for each calibrator is also shown.

FIG. 12 is a calibration curve showing the average fluorescenceintensity per pixel for calibrators of an analyte (i.e., neutrophilgelatinase-associated lipocalin, alternatively referred to herein as“NGAL”). For this calibration curve, the calibrators of the analyte NGALrange from 0 pM to 94 pM.

FIG. 13 is a calibration curve showing the average fluorescenceintensity per pixel for calibrators of an analyte (i.e., neutrophilgelatinase-associated lipocalin, alternatively referred to herein as“NGAL”). For this calibration curve, the calibrators of the analyte NGALrange from 0 pM to 6 pM.

FIG. 14 is a white light image of microparticles having complexes of anantibody:analyte:conjugate (i.e., monoclonal antibody19C7:troponin:conjugate M06-PE) attached thereto.

FIG. 15 is an image of a region of interest based on the image of FIG.14. FIG. 14 was generated by a software program.

FIG. 16 is a fluorescence image of microparticles having complexes ofantibody:analyte:conjugate (i.e., monoclonal antibody19C7:troponin:conjugate M06-PE) attached thereto.

FIG. 17 is a calibration curve generated from the value of averageintensity per pixel for each concentration of the calibrator troponin.

FIG. 18 is an illustration showing the protocol of an assay fordetecting DNA.

DETAILED DESCRIPTION

As used herein, the term “analyte” means a compound or composition to bemeasured, which may be a ligand, which is monoepitopic or polyepitopic,antigenic or haptenic, a single or plurality of compounds, which shareat least one common epitopic site or a receptor.

An immunoassay is a biochemical test that measures the presence orconcentration of a substance in solutions that frequently contain acomplex mixture of substances. Analytes in biological liquids such asserum or urine are frequently assayed using immunoassay methods. Suchassays are based on the unique ability of an antibody to bind with highspecificity to one or a very limited group of molecules. A molecule thatbinds to an antibody is called an antigen. Immunoassays can be carriedout for either member of an antigen/antibody pair. In addition tobinding specificity, the other key feature of all immunoassays is ameans to produce a measurable signal in response to a specific binding.Historically this was accomplished by measuring a change in somephysical characteristic such as light scattering or changes inrefractive index. Nevertheless most immunoassays today depend on the useof an analytical reagent that is associated with a detectable label. Alarge variety of labels have been demonstrated including enzymes;fluorescent, phosphorescent, and chemiluminescent dyes; latex andmagnetic particles; dye crystallites, gold, silver, and seleniumcolloidal particles; metal chelates; coenzymes; electroactive groups;oligonucleotides, stable radicals, and others. Such labels serve fordetection and quantitation of binding events either after separatingfree and bound labeled reagents or by designing the system in such a waythat a binding event effects a change in the signal produced by thelabel. Immunoassays requiring a separation step, often called separationimmunoassays or heterogeneous immunoassays, are popular because they areeasy to design, but they frequently require multiple steps includingcareful washing of a surface onto which the labeled reagent has bound.Immunoassays in which the signal is affected by binding can often be runwithout a separation step. Such assays can frequently be carried outsimply by mixing the reagents and sample and making a physicalmeasurement. Such assays are called homogenous immunoassays or lessfrequently non-separation immunoassays.

As used herein, the expression “sandwich immunoassay” means animmunoassay that employs at least two receptors that specifically bindto the same ligand. In this type of immunoassay, the ligand is theanalyte. One of the receptors is capable of specifically binding to theligand, whereby the receptor enables the ligand to be attached directlyor indirectly to a solid support, such as, for example, a microparticle.The other receptor is capable of specifically binding to the ligand,whereby the receptor enables the ligand to be attached directly orindirectly to a label to provide a signal for detecting the ligand. Forexample, one of the receptors can be a capture antibody for specificallybinding to an antigen in a sample, whereby the antigen is attacheddirectly or indirectly to a solid support, such as, for example, amicroparticle, and the other receptor can be a detection antibody forspecifically binding to the antigen in the sample, whereby the antigenis attached directly or indirectly to a label for detecting the antigen.If a relatively high amount of ligand is present in the sample, a highersignal will be produced. If a relatively low amount of ligand is presentin the sample, a lower signal will be produced. FIG. 1 is a schematicdiagram illustrating a representative example of a sandwich immunoassay.

As used herein, the expression “competitive immunoassay” means animmunoassay that employs a receptor that binds to a ligand. In this typeof immunoassay, the ligand is the analyte. The receptor is capable ofspecifically binding to the ligand, whereby the ligand is attacheddirectly or indirectly to a solid support, such as, for example, amicroparticle. A labeled ligand competes for the same receptor as doesthe analyte. For example, the receptor can be a capture antibody forspecifically binding to an antigen in a sample, whereby the antigen isattached directly or indirectly to a solid support, such as, forexample, a microparticle. The antigen in the sample is unlabeled. Alabeled antigen competes for the same capture antibody as does theunlabeled antigen. The labeled antigen that becomes attached to thesolid support provides a label for detecting the antigen. Alternatively,in the case where the receptor is an antigen for specifically binding toan antibody in a sample, whereby the antibody is attached directly orindirectly to a solid support, such as, for example, a microparticle, anunlabeled antibody and a labeled antibody can compete for the sameantigen. The labeled antibody that becomes attached to the solid supportprovides a label for detecting the antibody. If a relatively high amountof ligand is present in the sample, a lower signal will be produced. Ifa relatively low amount of ligand is present in the sample, a highersignal will be produced. FIG. 2 is a schematic diagram illustrating arepresentative example of a competitive immunoassay.

As used herein, the term “complex” means at least two molecules that arespecifically bound to one another. Examples of complexes include, butare not limited to, a ligand bound to a receptor, a ligand bound to aplurality of receptors, e.g., a ligand bound to two receptors, areceptor bound to a plurality of ligands, e.g., a receptor bound to twoligands.

As used herein, the expression “solid phase” means that state of areceptor wherein the receptor is attached to a surface of a solidsupport such that the receptor cannot break free from the solid supportin a liquid medium. A solid phase can easily be separated from a liquidin which the solid phase is dispersed. An example of a solid support towhich a receptor can be attached is a microparticle, such as, forexample, a magnetic microparticle. The microparticle can easily beseparated from a liquid in which it is dispersed. The microparticle isreadily dispersed in an aqueous medium.

As used herein, the expression “solid support” means a substance that isinstrumental in creating a solid phase. Representative examples of solidsupports, include but are not limited to, microparticles, micro-wells ofmicro-well plates, nanoparticles, gels, colloids, biological cells.

As used herein, the expression “capture antibody” means an antibody thatbinds an analyte, i.e., an antigen, to a solid support, with the resultthat the antibody attaches the analyte to the solid support, whereby theanalyte is attached to the solid support either directly or indirectlythrough an intervening moiety or intervening molecule.

As used herein the expression “detection antibody” means an antibodythat is attached to a moiety or to a molecule that provides or can bemade to provide a detectable signal in a chemical or biologicalreaction.

As used herein, the expression “specific binding pair” means twodifferent molecules, where one of the molecules has an area on thesurface or in a cavity which specifically binds to a particular spatialorganization of the other molecule. The members of the specific bindingpair are referred to as ligand and receptor.

As used herein, the expression “non-specific binding” means bindingbetween two or more entities, such as, for example, two molecules, in amanner other than that which results in a specific binding pair.

As used herein, the term “ligand” means any substance for which areceptor naturally exists or can be prepared. Such substances include,but are not limited to, organic compounds, inorganic compounds, andchemical elements, e.g., copper, lithium.

As used herein, the term “receptor” means any compound or compositioncapable of recognizing a particular spatial and polar organization of amolecule, i.e., epitopic site. Illustrative receptors include naturallyoccurring receptors, e.g., thyroxine binding globulin, antibodies,enzymes, Fab fragments, lectins, and the like.

Binding of a ligand to a receptor involves non-covalent interactionbetween two molecular species. Typically, but not necessarily, a smallerligand is a soluble molecule that binds to a larger receptor. Examplesof binding of a ligand to a receptor include, but are not limited to,the following:

-   -   (a) a long single strand DNA receptor having a complementary        sequence for a short single strand DNA ligand;    -   (b) any antibody receptor that binds to its complementary        antigen ligand or antigen receptor that binds to its        complementary antibody ligand;    -   (c) the intrinsic factor protein receptor that binds the vitamin        B₁₂ ligand;    -   (d) the hemoglobin receptor for the oxygen molecule ligand.

As used herein, the term “conjugate” means an entity comprising abinding pair member and a member of a signal producing system, e.g., alabel. As used herein, the term “label” means a member of a signalproducing system which is directly or indirectly bonded to a bindingpair member or to a microparticle. As used herein, the expression“signal producing system” refers to a system having one or morecomponents, at least one component being conjugated to a specificbinding pair member. The signal producing system produces a measurablesignal which is detectable by external means, usually the measurement ofelectromagnetic radiation, and depending on the system employed, thelevel of signal will vary to the extent the signal producing system isin the environment of the solid support, e.g., a microparticle. For themost part, the signal producing system will involve chromophores, wherechromophores include dyes which absorb light in the ultraviolet orvisible region, phosphors, fluorescers, fluorophores, luminophores, andchemiluminescers. In addition, enzymes can be employed to produce asignal or to amplify a signal or both of the foregoing.

As used herein, the terms “qualify”, qualified”, and the like refer to aprocedure for removing those signals from an image that are notattributable to complex comprising the analyte of interest. Such signalsthat are removed include, but are not limited to, signals arising fromnon-specific binding, undesired aggregation, or signals that emanatefrom locations that are not attached to the solid support. In mostcases, but with a few exceptions, those signals that qualify foranalysis and measurement are the result of specific binding of aconjugate that comprises a label.

As used herein, and in the field of digital imaging in general, the term“pixel”, or “pel” (picture element), means a single point in a digitalimage, or the smallest addressable screen element in a display device.It is the smallest unit of picture that can be represented orcontrolled. Each pixel has its own address. The address of a pixelcorresponds to its spatial coordinates. Pixels are usually arranged in atwo-dimensional grid, and are often represented using dots or squares.Each pixel is a sample of an original image; more samples typicallyprovide more accurate representations of the original. The intensity ofeach pixel is variable. The total number of pixels in an image can vary.A representative example of the number of pixels in a digital image is1024×1024.

As used herein, the term “intensity” means the amount or degree ofstrength of electricity, light, heat, or sound per unit area or volume.More particularly, with respect to the method actually described herein,the term “intensity” refers to the number of photons counted per unit ofarea per unit of time. For example, 1000 photons per unit area may berecorded as 500 counts in a single pixel, while 80 photons per unit areaare recorded as 40 counts in a single pixel. The particular conversiondepends on the camera system used. Intensity is proportional to thenumber of photons counted.

As used herein, the expression “region of interest” refers to thosepixels in an image that are selected for further analysis. In an image,pixels can be contiguous or non-contiguous.

As used herein, the phrase “spatial information” means identification ofa location from which a signal emanates.

As used herein, the term “IgG” means Immunoglobulin G.

In a general statement of the invention described herein, a ligandspecifically binds to a receptor to form a ligand-receptor complex. Animage is obtained of the ligand-receptor complex. The image ispreferably stored so that it can be used at a later time, if desired. Aregion of interest is selected from the image in such a manner that onlythe ligand-receptor complexes are studied further. This selectionfeature ensures that background signals and non-specific binding signalsare substantially eliminated from further analysis. The average numberof counts of light per pixel in the region of interest is calculated.

Ligands that are amenable to the method described herein include, butare not limited to, those mentioned in U.S. Pat. No. 4,275,149,incorporated herein by reference. Receptors that are amenable to themethod described herein include, but are not limited to, those mentionedin U.S. Pat. No. 4,275,149, incorporated herein by reference.Ligand-receptor complexes that are amenable to the method describedherein include, but are not limited to, those mentioned in U.S. Pat. No.4,275,149, incorporated herein by reference.

Receptors are typically attached to a solid support. A solid supportsuitable for use herein is a microparticle. Microparticles that aresuitable for use with the method described herein include, but are notlimited to, magnetic microparticles. The sizes of microparticlestypically range from about 0.1 μm to about 100 μm. Commerciallyavailable microparticles are available in a wide variety of materials,including those made of ceramics, glass, polymers, and metals. Magneticmicroparticle suitable for use in the method described herein arecommercially available from Polymer Laboratories, a subsidiary ofAgilent Technologies. Although the generally accepted definition of 0.1μm to 100 μm complements the size definition of nanoparticles, there areother ways to define the size. General acceptance considersmicroparticles smaller than 100 nm to be nanoparticles. Anymicroparticle larger than 0.5 μm and anything smaller than 0.5 mm isconsidered to be a microparticle. In general, the size of microparticlessuitable for use with the method described herein must be sufficientlylarge so that two microparticles can be resolved by the image systemselected. The properties of the microparticles suitable for use with themethod described herein, such as, for example, color, is a matter ofchoice. One of ordinary skill in the art can select the properties ofthe microparticles in order to fulfill requirements imposed byappropriate variations of the method.

Reaction vessels that are suitable for use with the method describedherein include micro-well plates. It is preferred that the reactionvessel be of such a character that an image of the ligand-receptorcomplex can be made. It is preferred that the reaction vessel betransparent to electromagnetic radiation, typically in the ultravioletand the visible range of the spectrum. Materials that are suitable formaking a reaction vessel include glass, and polymeric materials. It ispreferred that the material of the reaction vessel not beauto-fluorescent. However, the particular form or shape of the reactionvessel is not critical.

Reaction conditions for assays contemplated for use with the methoddescribed herein are not critical. Substantially the same conditionsthat are used with conventional immunoassays or other conventionalspecific binding reactions can be used. Such conditions include, but arenot limited to duration of incubation, temperature range of incubation,number of washing steps, buffers and other non-reactive substances inthe assays, and the like. In principle, any immunoassay designed for useas a chemiluminescent assay can be carried out by the method describedherein through the use of a fluorescent label.

Imaging systems suitable for use in the method described herein can beany system capable of acquiring images such that individualmicroparticles can be resolved. Imaging devices suitable for use withthe method described herein include, but are not limited to, lightmicroscopes, scanning microscopes, fluorescence imaging scanners, andthe like. Image file types that are suitable for use with the methoddescribed herein include, but are not limited to, JPEG/JFIF, GIF, BMP,TIFF, and FITS. Image file formats are described at Image fileformats—Wikipedia, the free encyclopedia, which is accessible by meansof Hypertext Transfer Protocol at the websiteen.wikipedia.org/wiki/image_file_formats, incorporated herein byreference, and FITS is described at FITS—Wikipedia, the freeencyclopedia, which is accessible by means of Hypertext TransferProtocol at the website en.wikipedia.org/wiki/FITS, incorporated hereinby reference.

Duration of exposure during acquisition of the image is not critical.Exposure times suitable for use with the method described herein can beany exposure time that provides sufficient resolution for discerningrelevant details of the image.

The selection of the region of interest is important. Through the use ofa suitable computer program, the locations of individual microparticlesare determined by means of contrast or some alternative criteria. Thepixels associated with the microparticles or other solid support can bedeemed a region of interest. In order to obtain a meaningful value ofconcentration of an analyte in a sample, it is preferred that at leastabout 100 microparticles, more preferably at least about 200microparticles be located in an image. Commercially available computerprograms suitable for use in the method described herein include, butare not limited to, those programs having the trademarks “SLIDEBOOK” and“METAMORPH” or software in the public domain, such as, for example,ImageJ.

In order to carry out a simplified form of the method, a commerciallyavailable epifluorescence microscope can be used to image the complexesthrough a transparent surface upon which they are supported. Arepresentative example of such a microscope is a motorized invertedfluorescence microscope (OLYMPUS “IX81”) coupled with a high resolutionCCD camera (Hamamatsu Model C4742-80-12AG), which are commerciallyavailable from numerous sources.

In this basic form of the method, a single-color approach can be used toprovide greater sensitivity than a conventional immunoassay employing alight signal from the total volume of a reaction mixture.

This greater sensitivity can be evidenced by a plot of a linear functionhaving a greater slope at lower concentrations relative to that of alinear plot employed as a calibration curve in a conventionalimmunoassay.

Microparticles bearing capture antibodies, detection antibodies attachedto fluorophores, and a sample suspected of containing an analyte arecombined under appropriate conditions to carry out an immunoassay. Afterthe immunoassay is carried out, any fluorescence signal that does notemanate from a complex comprising a microparticle attached to a captureantibody, an analyte, and a conjugate comprising a detection antibodyattached to a fluorophore is omitted. Then, the complexes remaining arefurther qualified based on fluorescence emitted by the fluorophore ofthe conjugate. This latter step omits any sections on the surface of themicroparticle that do not meet selection criteria. Based on astatistical parameter, such as, for example, standard deviation, atypical example of a selection criterion is that the microparticles tobe used for measurement have a substantially homogeneous coating, whichessentially eliminates excessive aggregation of conjugates, which canresult from a high degree of non-specific binding. In general, selectioncriteria vary, depending upon the particular assay. One of ordinaryskill in the art of the particular assay should be able to formulatemeaningful selection criteria for that particular assay. Finally, theaverage value of intensity per pixel of the qualified particles ismeasured in order to compare the intensity to a calibration curve thatestablishes concentration of the analyte as a function of intensity. Theaverage value of intensity per pixel of the qualified particles can bedetermined by means of a CCD camera, which is capable of measuringintensity of light. The measurement of intensity is converted to aparameter, which is designated in the units of counts. Each pixel has anumber corresponding to the intensity of light measured at that pixel.

In a preferred embodiment, a white light image of the reaction mixtureis obtained. The white light image is employed to locate the position ofeach solid support, e.g., a microparticle. A white light image is formedby using the entire electromagnetic spectrum for both illumination anddetection. This step is not required, but is preferred because it may bedesirable to locate the position of each solid support, e.g., amicroparticle. A fluorescence image is then acquired to determine thelocation and intensity of detection antibodies attached tomicroparticles. The fluorescence image uses a color, e.g., red, green.Counts per pixel are calculated and the average and standard deviationof counts per pixel are recorded. Pixels that have counts greater thanor less than, for example, two times the aforementioned standarddeviation are omitted from the analysis. The average number of countsper pixel for the pixels remaining are calculated. The quantity ofsignal measured from the label of the detection antibody determines theconcentration of the analyte.

In order to carry out a more sophisticated measurement that will providea higher degree of sensitivity, a commercially available epifluorescencemicroscope can be used to image the complexes through a transparentsurface upon which they are supported. A representative example of sucha microscope is a motorized inverted fluorescence microscope (OLYMPUS“IX81”) coupled with a high resolution CCD camera (Hamamatsu ModelC4742-80-12AG), which are commercially available from numerous sources.

In this more sophisticated measurement, a dual-color approach is used toprovide greater sensitivity than both a conventional immunoassayemploying a light signal from the total volume of reaction mixture and ameasurement made by the single-color approach described earlier. Thisgreater sensitivity is evidenced by a plot of a linear function having agreater slope at lower concentrations relative to that of a linear plotemployed as a calibration curve in a conventional immunoassay or anassay using the single-color approach.

Microparticles bearing capture antibodies, detection antibodies attachedto fluorophores, and a sample suspected of containing an analyte arecombined under appropriate conditions to carry out an immunoassay. Afterthe immunoassay is carried out, any fluorescence signal that does notemanate from a complex comprising microparticle attached to a captureantibody, an analyte, and a conjugate comprising a detection antibodyattached to a first fluorophore is omitted. Next, an image of thecapture antibody (characterized by a second fluorophore, which isdifferent from the first fluorophore) is obtained. This image omits anypixels corresponding to any microparticles that are not coated withcapture antibody in a homogeneous manner. If a microparticle is notuniformly coated, pixels from that portion that is not uniformly coatedare omitted. Then, the complex is further qualified based onfluorescence emitted by the conjugate. This latter step omits anysections on the complex that do not meet selection criteria. A typicalexample of a selection criterion is homogeneous coating, whichessentially eliminates excessive aggregation of conjugates, which canresult from a high degree of non-specific binding. As stated previously,selection criteria vary, depending upon the particular assay. Finally,the average value of intensity per pixel of the qualified particles ismeasured in order to compare the intensity to a calibration curve thatestablishes concentration of the analyte.

In a preferred embodiment, a white light image of the reaction mixtureis obtained. The white light image is employed to locate the position ofeach solid support, e.g., a microparticle. A white light image is formedby using the entire electromagnetic spectrum for both illumination anddetection. This step is not required, but is preferred because it may bedesirable to locate the position of each solid support, e.g., amicroparticle. A first fluorescence image is then acquired to determinethe locations of the capture antibodies attached to microparticles. Thefirst fluorescence image uses a color, e.g., red, green. A secondfluorescence image is acquired to determine the locations of antibodiesthat are present as a component of a conjugate. The second fluorescenceimage uses a color, e.g., red, green, but the color of the secondfluorescence image differs from the color of the first fluorescenceimage. Pixels derived from both a capture antibody on a microparticleand an antibody on a conjugate are selected for further analysis. Countsper pixel are calculated and the average and standard deviation ofcounts per pixel are recorded. Pixels that have counts greater than orless than, for example, two times the aforementioned standard deviationare omitted from the analysis. The average number of counts per pixelfor the pixels remaining are calculated. The quantity of signal measuredfrom the label of the detection antibody determines the concentration ofthe analyte.

FIGS. 3, 4, and 5 illustrate the steps required to qualify the resultsof a ligand-receptor binding assay. A fluorescence channel is definedwith a set of filters comprising an excitation filter and an emissionfilter, which allows light having a specific wavelength to reach thesample and a signal having a specific wavelength to reach the CCDcamera. For example, the fluorophore R-phycoerythrin (alternativelyreferred to herein as “PE”) can only be detected in the PE channel andcannot be detected in any other fluorescence channel. Similarly, thefluorophore indodicarbocyanine (alternatively referred to herein as“Cy5”) can only be detected in the Cy5 channel and cannot be detected inany other fluorescence channel. In FIG. 3, one channel of the detectormeasures the fluorescence image of a capture antibody, which was labeledwith red fluorophores (Cy5 channel). Only microparticles that have beencoated with capture antibody having the red fluorescent label appear.There are two locations (white circles) that may be omitted because theyare not attached to the microparticle. In FIG. 4, another channel of thedetector measures the fluorescence image of a detection antibodyattached to the microparticle. A very bright spot that is not consistentwith the intensity profile of the other microparticles appears and isdesignated with a white circle. This location may also be omitted fromthe analysis. FIG. 5 indicates the region of interest from which valuesof intensity per pixel can be calculated. Areas that did not meet givenselection criteria can be omitted from this type of analysis.

The following non-limiting examples further illustrate embodiments ofthis invention. In the following examples, all concentrations are byweight (w/w) unless otherwise indicated. In the following examples,conjugates were prepared by conventional means known to those ofordinary skill in the art, unless otherwise indicated. In EXAMPLE 1,unless otherwise indicated, microparticles that bear a coating ofanti-troponin monoclonal antibody 19C7 but which have not yet reacted inan immunoassay are referred to as “microparticles coated withanti-troponin monoclonal antibody 19C7”; microparticles that havereacted in an immunoassay and that are present in a sandwich complex arereferred to as “microparticles attached to monoclonal antibody19C7:troponin:conjugate M06-PE complex.” In EXAMPLE 2, unless otherwiseindicated, microparticles that bear a coating of anti-human IgGmonoclonal antibody but which have not yet reacted in an immunoassay arereferred to as “microparticles coated with anti-human IgG monoclonalantibody”; microparticles that have reacted in an immunoassay and thatare present in a sandwich complex are referred to as “microparticlesattached to conjugate 2322-Cy5:NGAL:conjugate 903-PE complex.”

EXAMPLE 1

This example illustrates an immunoassay for troponin, through the use ofa single fluorescent dye as the label for the detection antibody.

Microparticles coated with anti-troponin monoclonal antibody 19C7 wereprepared. A set of calibrators for troponin (TnI (28-110aa)-TnC) wereprepared at the following concentration in phosphate buffered saline(PBS) containing bovine serum albumin (BSA) (0.5%), surface active agent(“STANDAPOL”, 0.2%), and antimicrobial agent (“PROCLIN” 300, 0.1%). Thefollowing table lists the concentrations of Tn (28-110aa)-TnC in eachcalibrator.

TABLE 1 Concentration of TnI (28-110aa)- TnC (pg/mL) Calibrator A 0Calibrator B 10 Calibrator C 100 Calibrator D 500 Calibrator E 10,000Calibrator F 50,000 Low Control 20 Medium Control 200 High Control15,000A conjugate comprising anti-troponin monoclonal antibody M06 andphycoerithrin (PE) was prepared by means of a R-PE conjugation kit (PJ31K, Prozyme Inc.) according to the protocol suggested therein. Theconjugate is referred to herein as “conjugate M06-PE”.

R-Phycoerythrin, or PE, is useful in the laboratory as afluorescence-based indicator for labeling antibodies or other moleculesin a variety of applications. R-Phycoerythrin absorbs strongly at about566 nm with secondary peaks at 496 and 545 nm and emits strongly at 575nm. R-Phycoerythrin is among the brightest fluorescent dyes everidentified. See, for example, Phycoerythrin—Wikipedia, the freeencyclopedia, which is accessible by means of Hypertext TransferProtocol at the website en.wikipedia.org/wiki/Phycoerythrin andR-PHYCOERYTHRIN (PB31), ProZyme Inc., Hayward, Calif., both of which areincorporated herein by reference.

Each calibrator (100 μL) was mixed with microparticles coated withanti-troponin monoclonal antibody 19C7 (2.5 μL, 0.1%) and the conjugateM06-PE (2 μL, 68 nM) in a 96 micro-well glass-bottom plate for 15minutes at room temperature. The glass-bottom plate was used to reducethe level of auto-fluorescence. The 96 micro-well glass-bottom plate wasthen placed on a magnet (“DYNAL” “MPC”-96B) to attract themicroparticles attached to monoclonal antibody 19C7:troponin:conjugateM06-PE complexes to the bottoms of the micro-wells during the wash step.In the wash step, phosphate buffered saline (100 μL) was added to eachmicro-well and then quickly removed. This step was repeated twice.Phosphate buffered saline (50 μL) was added to each micro-well after thefinal wash step, and the plate was placed on a motorized invertedfluorescence microscope (OLYMPUS “IX81”) coupled with a high resolutionCCD camera (Hamamatsu Model C4742-80-12AG). After the microparticlesattached to monoclonal antibody 19C7:troponin:conjugate M06-PE complexessettled to the bottoms of the micro-wells, images of the microparticlesattached to monoclonal antibody-antigen-monoclonal antibody complexeswere taken with a UPlanSApo 20× objective (OLYMPUS) in the white lightchannel and the channel for the PE fluorophore. The viewing area of eachimage was approximately 400 micrometers×300 micrometers and usually hadapproximately 100 microparticles attached to monoclonal antibody19C7:troponin:conjugate M06-PE complexes. A plurality of locationswithin a given micro-well was imaged to improve statistical analysis.

FIG. 6 shows white light images of the microparticles attached tomonoclonal antibody 19C7:troponin:conjugate M06-PE complexes. FIG. 7shows fluorescence images of the microparticles attached to monoclonalantibody 19C7:troponin:conjugate M06-PE complexes. The white light imagewas used to locate the position of each individual microparticleattached to a monoclonal antibody 19C7:troponin:conjugate M06-PEcomplex, based on the contrast of the individual microparticles attachedto the monoclonal antibody 19C7:troponin:conjugate M06-PE complexes withthe background. FIG. 8 shows the locations of individual microparticlesattached to monoclonal antibody 19C7:troponin:conjugate M06-PEcomplexes, which were defined as the region of interest. The region ofinterest comprises the light spots in FIG. 8. The average value offluorescence intensity per pixel in the region of interest was thencalculated using the digital image in the PE channel. In FIG. 9, therewere spots substantially smaller than microparticles that did notoverlap the region of interest. These spots of high fluorescenceintensity emanated from the conjugates M06-PE that were not specificallybound to the surface of the micro-well plate. These spots were excludedfrom the analysis. The imaging and analysis approach described hereingreatly reduced the background signals that did not emanate from themicroparticles attached to monoclonal antibody 19C7:troponin:conjugateM06-PE complexes. The intensity values from all the calibrators wereused to generate a calibration curve. On account of the limited dynamicrange of the detector, a shorter exposure time was used for calibratorswherein concentrations of analyte are higher. FIG. 10 shows acalibration curve of calibrators ranging from 10 pg/mL to 50,000 pg/mL.FIG. 11 shows a zoomed-in calibration curve of calibrators ranging from0 pg/mL to 200 pg/mL. In the zoomed-in graph, the average value offluorescence intensity per pixel from the total area of the image foreach calibrator was also plotted for comparison. It is evident that theuse of spatial information of the image greatly increased the slope ofthe curve, thereby improving sensitivity of the immunoassay.

EXAMPLE 2

This example illustrates an immunoassay for neutrophilgelatinase-associated lipocalin (alternatively referred to herein as“NGAL”), through the use of two fluorescent dyes, one as a label for thecapture antibody, and the other as a label for the detection antibody.

Microparticles coated with anti-human IgG monoclonal antibody wereprepared. A set of calibrators for neutrophil gelatinase-associatedlipocalin ranging from 94 pM to 0.7 pM was prepared using HBS-EP buffer.

A conjugate comprising anti-NGAL monoclonal antibody 2322 and afluorescent dye (Cy5) and a conjugate comprising anti-NGAL monoclonalantibody 903 and R-Phycoerythrin (PE) were prepared. Monoclonal antibody2322 labeled with the Cy5 fluorophore is referred to herein as“conjugate 2322-Cy5”. Monoclonal antibody 903 labeled with the PEfluorophore is referred to herein as “conjugate 903-PE.”

Monoclonal antibody 2322 and monoclonal antibody 903 are monoclonalantibodies that can specifically bind to NGAL. Monoclonal antibody 2322is a human chimeric antibody, and monoclonal antibody 903 is a mouseantibody. The microparticles coated with anti-human IgG monoclonalantibody can bind directly to monoclonal antibody 2322.

Cy5 is a reactive water-soluble fluorescent dye of the cyanine dyefamily. Cy5 is fluorescent in the red region of the electromagneticspectrum (approximately 650 nm or 670 nm) but absorbs in the orangeregion of the electromagnetic spectrum (approximately 649 nm). Cy5 isalso used to label proteins and nucleic acid for various studiesincluding proteomics and RNA localization. See, for example, “TechnicalInformation on Probes Conjugated to Affinity-Purified Antibodies and toOther Proteins: Cyanine Dyes (Cy2, Cy3, and Cy5)”, which is accessibleby means of Hypertext Transfer Protocol on the World Wide Web at thewebsite jacsonimmuno.com/technical/f-cy3-5.asp, incorporated herein byreference.

The microparticles coated with anti-human IgG monoclonal antibody wereblocked with monoclonal antibody 903. Because monoclonal antibody 903 isa mouse monoclonal antibody, it should not cross-react withmicroparticles coated with anti-human IgG monoclonal antibody. However,some cross-reactivity was found when the microparticles coated withanti-human IgG monoclonal antibody were incubated with monoclonalantibody 903 labeled with a PE fluorophore. If the microparticles coatedwith anti-human IgG monoclonal antibody are first treated withmonoclonal antibody 903, and then reacted with monoclonal antibody 903labeled with a PE fluorophore, the cross-reactivity of microparticlescoated with anti-human IgG monoclonal antibody with monoclonal antibody903 labeled with a PE fluorophore is greatly reduced. The average valueof fluorescence intensity per pixel calculated from the region ofinterest in the images was 336 for microparticles coated with anti-humanIgG monoclonal antibody when treated with conjugate 903-PE and 136 formicroparticles coated with anti-human IgG monoclonal antibody firsttreated with monoclonal antibody 903 and then reacted with conjugate903-PE. Additional runs showed that treating the microparticles coatedwith anti-human IgG monoclonal antibody with monoclonal antibody 903does not decrease the signal generated from the analyte NGAL. When theconcentration of NGAL is relatively high, the effect of the signal inthe background of the image is not significant.

TABLE 2 shows fluorescence intensity of a sample containing 0 pM NGALusing (a) microparticles coated with anti-human IgG monoclonal antibodythat were treated with monoclonal antibody 903 and (b) untreatedmicroparticles coated with anti-human IgG monoclonal antibody. Theintensity of the treated microparticles coated with anti-human IgGmonoclonal antibody is approximately 40% that of the untreatedmicroparticles coated with anti-human IgG monoclonal antibody.

TABLE 2 No NGAL in sample Average value of intensity Average value ofintensity per pixel for microparticles per pixel for microparticlescoated with anti-human coated with anti-human IgG monoclonal antibodyIgG monoclonal antibody and treated with but untreated with monoclonalantibody 903 monoclonal antibody 903 (counts per pixel) (counts perpixel) Image 1 121.5 302.8 Image 2 125.4 320.0 Image 3 143.4 350.6 Image4 154.3 354.3 Image 5 136.2 355.0 Average 136.2* 336.5* Standard 13.323.8 deviation *dark counts from the camera are subtracted from theintensity

TABLE 3 shows the fluorescence intensity measured for a samplecontaining 300 pM NGAL using the microparticles coated with anti-humanIgG monoclonal antibody that were treated with monoclonal antibody 903and fluorescence intensity measured for a sample containing 300 pM NGALusing untreated microparticles coated with anti-human IgG monoclonalantibody. The immunoassay carried out for the purpose of determining theefficacy of using monoclonal antibody 903 as a blocking agent wasconducted in the same manner as the immunoassays that were conducted toverify the concentration of NGAL. Images were measured with a shorterexposure time than that used for the images characterized in TABLE 1.

TABLE 3 300 pM NGAL in sample Average value of intensity Average valueof intensity per pixel for microparticles per pixel for microparticlescoated with anti-human coated with anti-human IgG monoclonal antibodyIgG monoclonal antibody and treated with but untreated with monoclonalantibody 903 monoclonal antibody 903 (counts per pixel) (counts perpixel) Image 1 1491.8 1463.6 Image 2 1513.7 1467.0 Image 3 1520.5 1493.2Image 4 1521.2 1495.1 Image 5 1535.6 1538.4 Average 1516.6 1491.4Standard 16.0 30.0 deviation

Microparticles coated with anti-human IgG monoclonal antibody (1 mL,0.01%) were incubated with monoclonal antibody 903 (70 nM) for 15minutes. The thus-treated microparticles coated with anti-human IgGmonoclonal antibody were separated from the reaction mixture by means ofa magnet and then washed with HBS-EP buffer. The thus-treatedmicroparticles coated with anti-human IgG monoclonal antibody were thenreconstituted to a concentration of 0.01% with HBS-EP buffer. In thefirst row of micro-wells of a micro-well plate having 96 micro-wells,various concentrations of NGAL (94 pM, 47 pM, 23 pM, 12 pM, 6 pM, 3 pM,1.5 pM, 0.7 pM, 0 pM) were prepared. Each micro-well contained 100 μL ofliquid. Monoclonal antibody 2322 labeled with Cy5 fluorophore (25 μL, 2nM) and monoclonal antibody 903 labeled with PE fluorophore (8 μL, 20nM) were added to the first row of micro-wells in the plate. Thereaction mixtures were incubated for 15 minutes at room temperature.Microparticles coated with anti-human IgG monoclonal antibody (25 μL,0.01%) were added to each micro-well in the first row. The reactionmixtures were incubated for an additional 20 minutes. The microparticlesattached to conjugate 2322-Cy5:NGAL:conjugate 903-PE complexes wereattracted to a magnet and then washed three times with HBS-EP buffer.The samples were transferred to a micro-well plate in which themicro-wells had glass bottoms. The micro-well plate was placed on thefluorescence microscope (OLYMPUS “IX81”) and images were taken in thesame manner as described in EXAMPLE 1 in the white light channel, in thePE channel, and in the Cy5 channel.

The white light image was used to locate the positions of individualmicroparticles attached to conjugate 2322-Cy5:NGAL:conjugate 903-PEcomplexes based on the contrast between the individual microparticlesattached to conjugate 2322-Cy5:NGAL:conjugate 903-PE complexes and thebackground. The locations of individual microparticles attached toconjugate 2322-Cy5:NGAL:conjugate 903-PE complexes were defined asregions of interest. The regions of interest were further analyzed bysetting a threshold in the Cy5 channel, whereby only those areas on themicroparticles attached to conjugate 2322-Cy5:NGAL:conjugate 903-PEcomplexes having monoclonal antibody 2322 labeled with Cy5 fluorophorespecifically bound to the microparticles attached to conjugate2322-Cy5:NGAL:conjugate 903-PE complexes are used for further analysis.The average value of fluorescence intensity per pixel of the region ofinterest in the PE channel was calculated by means of a commerciallyavailable computer program (“SLIDEBOOK”).

The following table lists the average value of intensity per pixel fromthe PE channel for each concentration of analyte at the correspondingduration of exposure. Three different durations of exposure were used toincrease the dynamic range of the assay.

TABLE 4 Average Standard Exposure intensity per deviation time Number ofConcentration pixel (counts (counts per (ms) microparticles of NGAL (pM)per pixel) pixel) 200 176 0 342 5 200 187 0.72 391 23 200 225 1.46 47720 200 275 2.92 613 23 200 323 5.85 885 38 200 401 11.7 1625 90 200 34223.4 2558 19 50 525 11.7 526 10 50 405 23.4 859 23 50 458 46.8 1294 9550 342 93.75 1931 52

FIG. 12 shows calibration curves for NGAL generated from the data inTABLE 4. FIG. 13 shows the calibration curve at extremely lowconcentrations of calibrator. Three different algorithms were used todemonstrate the benefits of the present invention. Algorithm 2 andalgorithm 3 greatly improved the sensitivity of the assay.

The data for Algorithm 1 appear as solid circles (●) in the graph. Theaverage value of intensity of the entire image in the PE channel wascalculated. No spatial information was used. This algorithm isequivalent to a measurement of total intensity.

The data for Algorithm 2 appear as solid squares (▪) in the graph. Allof the microparticles were selected as regions of interest using thewhite light image based on the contrast level. The dimensions of theregions of interest were reduced by setting a threshold in the PEchannel. The cutoff was selected arbitrarily and was equal to theaverage value of fluorescence intensity of the image from the PE channelplus or minus three standard deviations. However, other cutoff valuescould have been used.

The data for Algorithm 3 appear as open squares (□) in the graph. Anarea on the microparticles attached to monoclonalantibody:antigen:monoclonal antibody complexes having intensity above athreshold in the Cy5 channel was selected, and then the average value offluorescence intensity of the PE channel was calculated. Signalsgenerated by non-specific binding were reduced. The cutoff was selectedarbitrarily and was equal to the average value of fluorescence intensityof the image from the PE channel plus or minus three standarddeviations. However, other cutoff values could have been used.

EXAMPLE 3

This example illustrates a homogeneous immunoassay for troponin, throughthe use of a single fluorescent dye as the label for the detectionantibody. In some immunoassays, where the concentration of analyte is inthe range of ng/mL, it is possible to perform a homogeneous immunoassayusing the method described herein. Such immunoassays are carried outsimply by mixing the reagents and sample and making a physicalmeasurement. Homogeneous immunoassays are desirable because they areeasy to perform.

All reagents and imaging parameters used in this example were the sameas those used in EXAMPLE 1. Each calibrator (100 μL) was mixed withmicroparticles coated with anti-troponin monoclonal antibody 19C7 (2 μL,0.1%) and the conjugate M06-PE (5 μL, 20 nM) in a 96 micro-wellglass-bottom plate for 15 minutes at room temperature. The glass-bottomplate was used to reduce the level of auto-fluorescence. Then PBS (200μL) was added to each micro-well to lower the concentration of theconjugate and to reduce the fluorescence intensity of the background.The plate was placed on a motorized inverted fluorescence microscope(OLYMPUS “IX81”) coupled with a high resolution CCD camera (HamamatsuModel C4742-80-12AG). After the microparticles attached to monoclonalantibody 19C7:troponin:conjugate M06-PE complexes settled to the bottomsof the micro-wells, images of the those microparticles attached tomonoclonal antibody 19C7:troponin:conjugate M06-PE complexes were takenwith a UPlanSApo 20× objective (OLYMPUS) in the white light channel andthe PE channel. The white light image, shown in FIG. 14, was used tolocate the position of each individual microparticle attached to amonoclonal antibody 19C7:troponin:conjugate M06-PE complex, based on thecontrast of the individual microparticles attached to the monoclonalantibody 19C7:troponin:conjugate M06-PE complexes with the background.FIG. 15 shows the locations of individual microparticles attached tomonoclonal antibody 19C7:troponin:conjugate M06-PE complexes, whichlocations were defined as the region of interest. The average value offluorescence intensity per pixel in the region of interest was thencalculated using the digital image in the PE channel, as shown in FIG.16. Using the spatial information of the image made it possible toeliminate a washing step and simplify the assay to a one-stephomogeneous assay. FIG. 17 shows the calibration curve calculated inthis example. Although a standard epi-fluorescence microscope was usedin this example, a confocal or TIRF (total internal reflectionfluorescence) microscope is preferred, because this type of microscopehas better z-plane resolution, which can eliminate signals from abovethe focal plane where the microparticles are positioned, therebylowering the background signal. A dilution step (adding buffer directlyto the reaction mixture) right before the measurement can further reducethe fluorescence background from the excessive antibody conjugates, andthus improve the sensitivity of assays.

Another approach for eliminating the wash step in the immunoassayinvolves analysis by image correlation spectroscopy (ICS).Spatio-temporal image correlation spectroscopy (STICS) analysis is anextension of ICS where a series of images of the sample are acquired.These images include both spatial and temporal information; theconjugates bound to the microparticles via formation of a sandwichcomplex are immobile while the excess unbound conjugates are freelydiffusing in the solution. STICS analysis can separate the immobileconjugate population from the diffusing conjugate population. There areother extensions of ICS, such as k-space Image Correlation Spectroscopy(kICS), and cross-correlation versions of ICS (CCICS) as well.Furthermore, by performing image correlation spectroscopy (ICS) analysison the images, additional information about the sample can be acquired.For example, in diagnosing Alzheimer's disease, it is possible to useICS to quantify the size of the amyloid plaques in an image as well asremove molecules undergoing diffusion.

EXAMPLE 4

This example illustrates how the method described herein can be used todetect DNA. In this case, the analyte is a target sequence of DNA andthe receptor is the DNA strand complementary to the target sequence. Fordetection, a labeled DNA strand having a sequence identical to thetarget sequence is used. The principle of a competitive assay for a DNAtarget is shown in FIG. 18.

In this example, single-stranded DNA (ssDNA) immobilized onmicroparticles is mixed with a sample suspected of containing targetssDNA and incubated. The mixture also contains ssDNA identical to thetarget ssDNA but having a fluorescent label for determining theconcentration of DNA. The ssDNA that is attached to a fluorescent labelwill be blocked by the presence of the target ssDNA. Therefore, if nofluorescence is detected, it can be concluded that the sample contains asufficient amount of ssDNA to completely block the ssDNA having thefluorescent label. This condition would indicate a positive result. Onthe other hand, if there are no target ssDNA molecules in the sample,the ssDNA immobilized on the microparticles will be completely labeledwith ssDNA having a fluorescent label. The reaction mixture shown inFIG. 18 illustrates the case in which the sample does contain targetssDNA, but not enough to completely block binding of the ssDNA havingthe fluorescent label. Therefore, both target ssDNA and ssDNA having afluorescent label will be present and the fluorescence intensity willcorrespond inversely with the amount of target ssDNA in the sample.

Various modifications and alterations of this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention, and it should be understood that thisinvention is not to be unduly limited to the illustrative embodimentsset forth herein.

What is claimed is:
 1. A method for determining the concentration of aligand in a sample comprising the steps of: (a) combining in a reactionmixture (i) a sample suspected of containing a ligand, (ii) a firstreceptor attached to a microparticle that binds to the ligand, and (iii)a fluorescently labeled second receptor that binds to the ligand, andallowing formation of a complex comprising the microparticle attached tothe first receptor, the ligand, and the fluorescently labeled secondreceptor; (b) acquiring a white light image of the reaction mixture inorder to determine the location of the microparticle in the reaction ofstep (a) and a fluorescence image of the reaction mixture in order todetermine the location of the fluorescently labeled second receptor inthe reaction of step (a); (c) selecting at least one region of interestfrom the images acquired in step (b), wherein the at least one region ofinterest is a region from which light signals emanate from the complexformed in step (a); (d) selecting pixels in the at least one region ofinterest for analysis; (e) calculating and recording the average andvariance of the counts per pixel for the pixels selected in step (d),wherein the counts per pixel is the number of photons counted per pixelper unit of time; (f) omitting pixels that have counts greater or lessthan a specified variance; (g) calculating average counts per pixel ofthe remaining pixels; and (h) determining the concentration of theligand from the data in step (g).
 2. The method of claim 1, wherein theimages acquired in step (b) are digital images.
 3. The method of claim1, wherein only up to about two hundred (200) microparticles arerequired.
 4. The method of claim 1, wherein the images acquired in step(b) are recorded by a fluorescence microscope equipped with a digitalcamera.
 5. The method of claim 1, wherein a record of the assay isstored in computer data storage.
 6. The method of claim 1, wherein theimages acquired in step (b) are acquired and stored off-line.
 7. Themethod of claim 1, wherein spatial information is used to qualify andquantify the results of a ligand-receptor binding assay.
 8. The methodof claim 1, wherein the ligand is a single stranded nucleic acidsequence, the first receptor is a capture nucleic acid sequencecomplementary to the single stranded nucleic acid sequence, and thesecond receptor is a fluorescently labeled nucleic acid sequence that isidentical to the single-stranded nucleic acid sequence.
 9. The method ofclaim 1, wherein the omitted pixels of step (f) have counts greater orless than two times the variance.